A protein is a biopolymer which exerts a specific function through the formation of a definite high-order structure which will be called a "natural form" hereinafter. A disulfide bond between cysteine residues involved in the above-mentioned high-order structure plays an important role in the performance of the function of the protein or in stabilizing the same. Recent development in gene recombination techniques has increased the production of proteins. In an expression system using Escherichia coli, in particular, a protein in the form of inclusion bodies is produced within the cells of the microorganism [cf. F.A.O. Marston et al., BIO/TECHNOLOGY, 2, 800 (1984); and D. N. Brems et al., Biochemistry, 24, 7662 (1985)]. Thus, it is an important problem to collect the desired protein from these inclusion bodies. In order to solubilize these proteinaceous inclusion bodies, it is necessary to once denature the same. When a cysteine-containing protein is to be solubilized, it is necessary to denature this protein in a reduced state. Thus, disulfide bonds should be formed at the same sites as those observed in the corresponding natural protein in order to efficiently reactivate the reduced and denatured protein.
A conventional process for converting a denatured protein into a natural one in the presence of a denaturing agent is carried out by diluting or dialyzing the starting protein with a solution free from any denaturing agent via one or more steps to thereby reactivate the same [cf. J. A. Gill et al., BIO/TECHNOLOGY, 3, 643 (1985); H. J. George et al., DNA, 4, 273 (1985); and JP-A-61-257931 (the term "JP-A" as used herein means an "unexamined published Japanese patent application")], since this transformation would frequently proceed in two-states [cf. Tanpakushitsu Bunshi (Protein Molecules), 99-127, Iwanami Shoten (1985)]. In this process, secondary and tertiary structures of the protein are also formed at the same time. Thus, hydrophobic groups, which are enclosed in protein molecules in the natural protein, would interact with each other or intermolecular or intramolecular disulfide bonds would be formed at sites different from those observed in the natural one. As a result, not the desired natural protein but an associated or denatured one would be frequently obtained. In the case of a protein having a marked tendency to form an associated or denatured material through the interaction between hydrophobic groups, in particular, the formation of disulfide bonds at the same sites as those of the natural one is considerably suppressed and thus the desired natural protein is obtained sometimes at a yield as low as approximately 1%.
Therefore, it is difficult to efficiently reactivate a reduced and denatured cysteine-containing protein, which is liable to be converted into an associated or denatured one, by the interaction between hydrophobic groups by a conventional reactivating process.
K. E. Langley et al. reported that a protein can be reactivated by forming disulfide bonds in a denatured protein at the same sites as those of the corresponding natural protein and at a high frequency, compared with the case in which the formation of disulfide bonds at the same sites as those of the natural one is inhibited by the formation of an associated or denatured one through the interaction between hydrophobic groups.
The process for reactivating bovine growth hormone of K. E. Langley et al. comprises (1) washing proteinaceous inclusion bodies produced within the cells of Escherichia coli, followed by solubilizing the inclusion bodies in 6 M guanidine hydrochloride; (2) oxidizing the inclusion bodies by allowing to stand at room temperature for 20 hours or more so that the formation of disulfide bonds occurs; (3) subjecting to gel filtration in the presence of 6 M guanidine hydrochloride; (4) collecting the monomer-containing fractions; and (5) diluting the fractions to the extent that the guanidine hydrochloride concentration is 2 M, followed by dialysis [K. E. Langley et al., Eur. J. Biochem , 163, 313-321 (1987)]. However, this process has such a disadvantage that since no reducing agent is added at the solubilization of the inclusion bodies, yields of the reduced monomer are low and formation of disulfide bonds at the same sites as those of the natural one is inhibited due to the action of the associated materials of the desired protein and substances contaminating the inclusion bodies which undesirably block the SH groups of the desired protein. Also, during oxidation of the inclusion bodies by allowing to stand at room temperature for 20 hours or more, aspargine residues and glutamine residues are deamidated [Biochemica. et Biophysica. Acta., 214, 498-508 (1970)]. If the oxidation is carried out at a low temperature, i.e., about 5.degree. C., in order to prevent the aspargine and glutamine residues from deamidation, it would take a prolonged time for oxidation.